Reagents for isolation of purified RNA

ABSTRACT

Compositions and methods to isolate intact RNA that is substantially free of DNA, termed purified RNA. RNA from any source (e.g., human, other animals, plants, viruses, etc.) may be isolated. In one embodiment, the sample is treated with phenol at a pH less than 4.0 and purified RNA is recovered from the aqueous phase. In another embodiment, RNA is precipitated from an acidified sample containing a low volume of an organic solvent. Other embodiments are disclosed. The same inventive composition may be used for several embodiments with pH adjustment. Purified RNA obtained by the inventive method may be used in assays where DNA contamination is undesirable, such as the polymerase chain reaction.

RELATED APPLICATIONS

This application is a divisional of Ser. No. 10/826,113, filed Apr. 16,2004, which is incorporated in its entirety herein.

FIELD OF THE INVENTION

The invention is directed to compositions and methods that enhanceisolation of purified RNA from biological samples.

BACKGROUND

Isolation of pure, intact RNA is a critical step for analysis of geneexpression in molecular biology, clinical, and biotechnologyapplications. Methods of RNA isolation have been developed in an attemptto achieve this goal. The most frequently used methods for RNA isolationare based on phenol extraction, precipitation from chaotropic saltsolutions, and adsorption on silica (Ausubel et al, 2002), reviewed inmy U.S. Pat. Nos. 4,843,155; 5,346,994; and 5,945,515. The methoddescribed in the '155 patent is frequently referred to as thesingle-step method and extracts RNA with a phenol-guanidine solution atpH 4. Its effectiveness and simplicity make the single-step method themost frequently used method for isolating RNA.

An improvement of the single-step method, described in my subsequent'994 patent, allowed simultaneous isolation of RNA, DNA, and proteinsfrom the same sample by phenol-guanidine extraction at pH 4-6. Abiological sample is homogenized and the homogenate is subjected tophase separation using a hydrophobic organic solvent such as chloroformor bromochloropropane. Following centrifugation, the mixture separatesinto the top aqueous phase containing RNA, and the interphase andorganic phase containing DNA and proteins. The aqueous phase iscollected and RNA is precipitated and washed with alcohol.

In the single-step method described in the '155 and '994 patents, acareful collection of the separated aqueous phase is critical for thequality of the isolated RNA. Small amounts of the interphase and organicphase can be easily removed together with the aqueous phase, whichresults in contamination of the isolated RNA with DNA and proteins.Also, collection of the aqueous phase requires a manual approach, whichis an obstacle in adapting the single-step method for automation.

The reagents and methods described in the '155 and '994 patents providesubstantially pure, undegraded RNA. However, RNA isolated according tothe '155 and '994 patents contains a residual amount of genomic DNA,which can be detected by reverse transcription-polymerase chain reactionassay (RT-PCR). Thus, RNA isolated in accord with the '155 and '994patents must be further purified to render it DNA-free (Guan at al,2003; Girotti and Zingg, 2003). The contaminating genomic DNA serves asa matrix for DNA polymerase, yielding additional amplification productsand distorting RNA-dependent RT-PCR. The DNA contamination in RT-PCR canbe only partially alleviated by using a set of primers encompassingexon-intron sequences in the genomic DNA because the presence ofpseudogenes, containing no introns, makes this approach unreliable(Mutimer 1998).

Modifications to the single-step method have improved the quality of theisolated RNA. In one modification, RT-PCR inhibitors were removed byadding a lithium chloride precipitation step (Puissant, 1990; Mathy,1996). In another modification, alcohol precipitation of RNA in thepresence of salt increased purity of the isolated RNA (Chomczynski,1995). These modifications, however, were not effective in removing DNAcontamination.

A common practice for removing contaminating DNA is to treat anRNA-containing sample with deoxyribonuclease (DNase). Following DNasetreatment, the RNA-containing sample is extracted sequentially withphenol and chloroform. In an effort to limit DNA contamination, anadditional DNA precipitation step was included in the single-stepmethod. The contaminating DNA was precipitated from the aqueous phase byadding one-third the volume of 95%^(w/w) ethanol (Siebert, 1993). Thefinal concentration of ethanol was about 24%^(w/w). The author indicatedthat, at this low ethanol concentration, DNA was precipitated while RNAremained in solution. RNA was precipitated from the solution by addingadditional alcohol. This protocol, however, yielded RNA that was stillcontaminated with DNA, evidenced as a visible band upon analyzing theisolated RNA on an agarose gel stained with ethidium bromide and byRT-PCR.

In another effort to diminish DNA contamination and improve the qualityof RNA in the single-step method, Monstein (1995) in a laboriousprocedure increased the pH of the phenol extraction to pH 4.1-4.7 andtreated the sample with proteinase K, followed by another round ofphenol extraction, precipitation, and ethanol wash. Despite thisprolonged procedure, DNase treatment was still necessary to obtainDNA-free RNA ready for use in RT-PCR.

Separating RNA from DNA was also achieved by phenol extraction at pH 4without adding guanidine salts (Kedzierski, 1991). However, the absenceof guanidine salts during the procedure made RNA susceptible toribonuclease (RNase), thereby degrading the RNA. A later improvement ofthis protocol employed phenol extraction buffer at pH 4.2 in thepresence of sodium dodecyl sulfate (Chattopadhyay et al., 1993). DNasetreatment was also required in the RNA isolation method using acombination of the single-step method followed by the silica columnprocedure (Bonham, 1996). The use of this double purification protocoldecreased DNA contamination, but the isolated RNA still containedgenomic DNA that was detected by RT-PCR. Another method for isolatingRNA used a monophase aqueous solution containing 10%^(w/w to) 60%^(w/w)phenol (U.S. Patent Application Publication 20030204077). In the absenceof chaotropes, 15%^(w/w) to 55%^(w/w) monoalcohol, diol, or polyol wasused to keep phenol in aqueous solution

Thus, a residual amount of DNA present in RNA isolated by the methodsdescribed in the '155 and '994 patents made it necessary to extend theprocedure by including DNase treatment. This diminished the usefulnessof the methods by prolonging procedures and unnecessarily exposing RNAto the possibility of degradation during DNase treatment and additionalpurification steps. However, removing residual DNA from RNA preparationsis needed for RT-PCR based microarray determination of gene expression.

Previous methods for isolating RNA, as described in the '155 and '994patents, were based on phenol extraction performed at pH 4 or higher.None of the previous modifications of the single-step method attemptedto improve the quality of RNA by performing phenol extraction at a pHbelow 4. To the contrary, pH 4 as used in the first '155 patent wasincreased in the next '994 patent to a pH ranging from 4 to 6.Similarly, the protocol described by Monstein (1995) increased the pH ofthe phenol extraction to pH 4.7. Another elaborate attempt to improvethe single-step method increased the pH of the guanidine-phenol extractto pH 5.2 (Suzuki, 2003).

An alternative to the single-step method of RNA isolation was disclosedin U.S. Pat. No. 5,973,137, using non-chaotropic acidic salts. However,the single-step phenol extraction method is still the most frequentlyused method for RNA isolation. A publication describing the single-stepmethod (Chomczynski 1987) is the fourth most cited paper in the databaseof the American Chemical Society and Institute for ScientificInformation, and the most cited paper published within the last twentyyears (CAS 2003, American Chemical Society).

New methods to enhance purity of isolated RNA are thus desirable.

SUMMARY OF THE INVENTION

The present invention discloses reagents and methods capable ofisolating from a biological sample RNA that is substantially free of DNAand thus ready for reverse transcriptase polymerase chain reaction(RT-PCR). Such RNA is termed substantially pure RNA, and is required forproper diagnosis of gene expression in clinical, research and otherapplications.

One embodiment is a phase separation method using acidic phenol, withRNA separating in the aqueous phase. This is based on the unexpectedfinding that substantially pure RNA can be isolated by phenol extractionperformed at pH below 4.

Another embodiment is acidic phenol precipitation of DNA and protein,with RNA remaining in the soluble fraction. This is based on theunexpected finding that certain concentrations of acidic phenolselectively precipitate DNA, proteins and other cellular components,leaving RNA remaining in a soluble form. The use of acidic phenol forselectively precipitating DNA and proteins eliminates the need for phaseseparation and also eliminates the use of toxic phase-separationsolvents. This approach significantly simplifies the RNA isolationprocess.

Another embodiment is selective RNA precipitation from solutionscontaining phenol, a chaotrope, and a low volume of an organic solvent.This embodiment may be used to selectively precipitate RNA molecules upto about 200 nucleotides. Shorter RNA molecules (lower molecular weightRNA) and/or DNA may also be recovered. DNA may also be recovered fromthe sample by increasing the concentration of organic solvent to atleast about 50%^(w/w).

Another embodiment is RNA precipitation from solutions containing atleast one salt by adjusting the pH of the solution to a maximum pH of3.3.

RNA isolated by the inventive compositions and methods can be useddirectly for RT-PCR because it has a higher purity, that is, there isless contamination of RNA by DNA and/or protein, in comparison toprevious methods for RNA isolation such as methods disclosed in U.S.Pat. Nos. 4,843,155; 5,346,994; and 5,945,515, each of which isexpressly incorporated herein by reference in its entirety. The RNA thatis isolated may be single stranded (ssRNA) or double stranded (dsRNA),and may be isolated from a variety of biological sources, includinganimals, plants, yeasts, bacteria, and viruses. RNA isolated by theinventive methods and using the inventive compositions may be used inmolecular biology, biotechnology, and clinical sciences. In addition,the inventive reagents may be used alone or in combination with othermethods for isolating substantially pure DNA (DNA substantially free ofRNA and protein), and substantially pure proteins (proteinssubstantially free of RNA and DNA).

These and other advantages will be apparent in light of the followingdetailed description and examples.

DETAILED DESCRIPTION

Methods and compositions to prepare purified RNA from biological samplesare disclosed. A biological sample is any sample from a biologicalsource, whether in vivo, in vitro, or ex vivo. Samples may be fromhumans, animals, plants, bacteria, viruses, fungi, parasites,mycoplasmas, etc. Purified RNA is RNA that is substantially undegradedand free of DNA contamination when assayed by reverse transcriptasepolymerase chain reaction (RT-PCR).

Phase Separation

One embodiment of the invention provides methods and reagents to enhancethe purity of isolated RNA by performing phenol extraction of anRNA-containing sample at a pH below 4.0. In one embodiment, the pHranges from about pH 3.9 to about pH 3.6. Phenol extraction at a pHbelow 4.0 more effectively separates RNA from DNA than phenol extractionat pH 4.0 or higher.

The RNA isolating reagent used in the inventive phase separation methodcomprises an aqueous solution of phenol, and a buffer to maintain the pHwithin the range from about 3.6 to below pH 4.0. In one embodiment, thepH ranges between pH 3.7 to pH 3.9. The effective concentration ofphenol in the RNA isolating reagent ranges from about 10%^(w/w) to about60%^(w/w). In one embodiment, the concentration of phenol ranges fromabout 25%^(w/w) to about 45%^(w/w).

The composition may also include other components, such as inhibitors ofribonuclease (RNase), salts, chelating agents, solubilizing agents,detergents, chaotropes, and phenol derivatives.

In some embodiments, RNA in samples having low RNase activity, such ascultured cells, may be extracted with acidic phenol at a pH betweenabout 3.6 to below pH 4.0, and this may sufficiently protect against RNAdegradation. However, phenol may not adequately prevent degradation ofRNA by cellular RNases derived from the sample or from contaminatedlabware. Thus, an effective amount of at least one RNase inhibitor maybe included in the composition. The RNase inhibitor may be presentduring sample homogenization and/or during acid phenol extraction. RNaseinhibitors include proteinase K, ribonuclease inhibitor from humanplacenta, vanadyl ribonucleoside complex, and chaotropic salts.Chaotropic salts include guanidine thiocyanate, guanidine hydrochloride,and mixtures of these. In one embodiment, an effective concentration ofchaotropic salts ranges from about 0.5 M to about 6 M. In anotherembodiment, an effective concentration of chaotropic salts ranges fromabout 2 M to about 4 M.

The buffer may be salts of at least one of acetate, citrate, phosphate,phthalate, tartrate, or lactate. The concentration of buffer should besufficient to maintain the composition at a pH between about 3.6 tobelow 4.0. In one embodiment, the pH ranges from about 3.75 to about3.85. The buffer may be added before or after sample homogenization,either separately or together with the phase separation reagent. Somesamples with a high buffering capacity, such as blood and plant tissues,may require an additional amount of acid to adjust the pH within thedesired range.

The inventive composition may also contain organic and inorganic saltssuch as chloride, phosphate, acetate and thiocyanate salts of sodium,potassium, lithium and ammonium. The inventive composition may containchelating agents such as citrates and ethylenediamine tetraacetatesalts. The inventive composition may contain detergents such aspolyoxyethylenesorbitan, sodium dodecylsulfate and sarcosine. The salts,chelating agents, and detergents promote tissue solubilization andprecipitation of substantially pure RNA. To assist in solubilizingphenol, the aqueous composition may contain a solubilizer or mix ofsolubilizers. Solubilizers include polyalcohols such as glycerol at aconcentration from about 1%^(w/w) to about 10%^(w/w), the upper limitselected so as not to increase DNA contamination of the isolated RNA.Solubilizers also include guanidine salts.

The inventive composition may contain within the about 60%^(w/w) phenol,up to about 5%^(w/w) of phenol derivatives that are less toxic thanphenol itself. These derivatives include phenylethanol, propylenephenoxytol, thymol, or butylphenol. In one embodiment these derivativesare present in an amount ranging between about 1%^(w/w) to about5%^(w/w). The composition may also contain insoluble or partiallywater-soluble organic compounds, such as cyclohexanol, cyclohexylbromide, and dichlorobenzoic acid. These compounds increase the densityof the composition and substitute for phenol, thereby minimizing thetoxicity of the composition.

In one embodiment of the phase separation method, a sample is prepared,typically by homogenization or lysis, in the inventive composition. Thebulk of DNA and particulate matter may be removed by sedimentation orfiltration from the homogenate or lysate. The homogenate or lysate isseparated into aqueous and organic phases by mixing with a hydrophobicorganic solvent or mix of solvents, such as chloroform, carbontetrachloride, bromonaphtalene, bromoanisole or bromochloropropane. Themixture may be sedimented by centrifugation, for example, centrifugationat a temperature in the range between about 4° C. to about 10° C. Thetop aqueous phase contains RNA, and the interphase and organic phasecontains DNA and proteins.

RNA is precipitated from the aqueous phase with a water-soluble organicsolvent, such as a lower alcohol. The precipitated RNA is washed bysedimentation or filtration and solubilized in water, formamide, or abuffer. The final RNA preparation is substantially pure, that is, it isundegraded and is essentially free of DNA contamination when tested byRT-PCR.

Additionally, the inventive phase separation method is compatible withthe method for the simultaneous isolation of RNA, DNA, and proteins. TheDNA and proteins sequestered into the interphase and organic phase maybe recovered, as described in Chomczynski, 1993; TRI Reagent brochure,2003. Alternatively, DNA is precipitated from the organic phase andinterphase by adding 0.3 volume of ethanol, followed by precipitation ofproteins with a higher amount of ethanol. For example, DNA can bere-extracted from the interphase and organic phase with an aqueoussolution at pH 7.0 or higher. Re-extracted DNA is precipitated from theaqueous solution with ethanol. As will be appreciated, the inventivecomposition and method may be used to isolate substantially pure RNA,substantially pure DNA (that is, DNA essentially free of RNA), andproteins from the same sample. Isolation of all three components allowsfor correlation of gene expression patterns with changes in the DNAsequence and protein content in biological samples, as well as havingnumerous other applications.

In one embodiment, the composition used for homogenizing or lysing thesample may lack one or more components, which would be thereafter addedto the homogenate or lysate, either alone or together with the phaseseparation solvent (for example chloroform). In another embodiment,sample homogenization or lysis may be performed above pH 4.0, in whichcase an acid or a buffer is then added to the homogenized or lysedsample in an amount sufficient to bring the pH of the homogenate orlysate within the range between about 3.6 to below pH 4.0. This amountof acid or buffer may be directly added to the homogenate or lysate, orit may be dissolved in the phase separation solvent. When added togetherwith the phase separation solvent, the acid may be formic acid, aceticacid, trichloroacetic acid, aminocaproic acid, lactic acid, orchlorophenylacetic acid. To promote acid solubility, the phaseseparation solvent may contain solubilizers such as glycols.

In one embodiment, sample homogenization or lysis is performed in aphenol-free solution containing an RNase inhibitor. After homogenizationor lysis, phenol is added to achieve a final concentration ranging fromabout 10%^(w/w) to about 60%^(w/w) and extraction is performed at pHfrom about 3.6 to below 4.0. This pH range during extraction ismaintained by a buffer that may be part of the aqueous solution, oradded to phenol, or may be added separately. After phase separation bycentrifugation, RNA is precipitated from the aqueous phase with alcohol.The precipitated RNA is washed and may be dissolved in a solvent such aswater, buffer or formamide.

Acidic Phenol Precipitation of DNA Leaving RNA in Supernatant

One embodiment of the invention isolates substantially pure RNA using anacidic phenol solution without performing phase separation. Certainconcentrations of acidic phenol selectively precipitate DNA (both singlestranded DNA (ssDNA) and double stranded DNA (dsDNA), proteins, andother cellular components, while RNA remains in a soluble form. Thisunexpected phenomenon was utilized to elaborate reagents and methods forisolating RNA without separating aqueous and organic phases and theinterphase.

The acidic phenol precipitation method simplifies the process of RNAisolation. It also eliminates toxic organic solvents that may be used inthe phase separation method. The composition propels DNA and proteins toform a firm pellet at the bottom of a tube, which alleviates the dangerof accidental transfer of DNA and protein molecules to the supernatantfraction containing RNA. The supernatant can be securely collected bypipetting, siphoning, decanting, or filtering, each of which may beautomated for use in an automated procedure for RNA isolation.Additionally, the entire acidic phenol precipitation method may occur atroom temperature, which eliminates the need for a refrigeratedcentrifuge that may be used in the phase separation method.

The composition used for acidic phenol precipitation comprises anaqueous solution of phenol at a concentration ranging from about3%^(w/w) to less than 30%^(w/w). In one embodiment, the phenolconcentration ranges from about 3%^(w/w) to about 25%^(w/w). In anotherembodiment, the phenol concentration is in the range between about8%^(w/w) to about 20%^(w/w). The phenol concentrations in the acidprecipitation embodiment are lower than the 30%^(w/w) phenol to60%^(w/w) phenol concentrations described in the '155 and '994 patents.

The inventive composition is acidified with a buffer or an acid in anamount sufficient to maintain the pH within a range from about 3.6 toabout 5.5. In one embodiment, the pH ranges from about 3.9 to about 4.5.The buffer can be selected from organic or inorganic buffers including,but not limited to, acetate, citrate, phosphate, phthalate, tartrate,and/or lactate.

To enhance the efficiency of RNA isolation, acidic phenol may besupplemented with RNase inhibitors, salts, chelating agents, phenolsolubilizing agents and/or detergents. RNase inhibitors include vanadylribonucleoside complex and protinase K or combinations of theseinhibitors. RNase inhibitors also include chaotropic agents orchaotropes such as guanidine salts at concentrations ranging from about0.5 M to about 6 M. In one embodiment, the concentration of thechaotropes is from about 1.5 M to about 2.5 M. Chaotropic salts mayserve as phenol solubilizers by maintaining phenol in aqueous solution.The acidic phenol composition may further contain organic and/orinorganic salts such as chloride, phosphate, acetate, citrate andthiocyanate salts of sodium, potassium, lithium, and ammonium. Thecomposition may also contain chelating agents such as citrates andethylenediamine tetraacetate salts. The composition may also containdetergents including polyoxyethylenesorbitan, sodium dodecylsulfate, andsarcosine. The composition may also contain up to 5%^(w/w) of solventsand reagents that are less toxic than phenol, such as thymol,phenylethanol, cyclohexanol, cyclohexyl bromide and dichlorobenzoicacid. These additional components promote tissue solubilization andprecipitation of pure RNA.

The acidic phenol precipitation reagent may contain an additionalsolubilizer or mix of solubilizers to help maintain phenol in aqueoussolution. Phenol solubilizing agents include glycols, polyalcohols, andlower alcohols. These can be added to the acidic phenol precipitationreagent in amounts from about 1%^(w/w) to about 10%^(w/w), the upperlimit selected so as not to increase DNA contamination of the isolatedRNA.

In one embodiment of the acidic phenol precipitation method, abiological sample is homogenized or lysed in the precipitation reagent.The resulted homogenate or lysate is centrifuged or filtered to removeprecipitated DNA, proteins, and other cellular components. RNA remainsin a soluble form and is subsequently precipitated from the supernatantwith a water-soluble organic solvent, such as lower alcohols includingmethanol, ethanol, propanol, isopropanol, and butanol. The RNA pellet isthen washed and may be dissolved in water, buffer, or formamide.

In the another embodiment of the acidic phenol precipitation method, abiological sample is homogenized in about 3 times (3×) to about 1.5times (1.5×) concentrated acidic phenol precipitation reagent. The useof a concentrated reagent allows processing of solid tissues as well ashigh volume liquid samples using one reagent. A high volume of a liquidsample can be compensated by adding to the concentrated reagent asmaller amount of water. The concentrated reagent dissolves most of thecomponents in a biological sample and effectively releases RNA fromcellular structures. For example, a sample may be homogenized in twotimes (2×) concentrated reagent. Following homogenization, an equalvolume of water is mixed with the homogenate. The addition of waterbrings phenol, guanidine, and other ingredients within the desiredconcentration. This creates conditions for effectively precipitating andremoving DNA and proteins from an RNA containing sample.

After centrifugation or filtration of the homogenate or lysate, RNAremains in the supernatant or filtrate, while DNA, protein, and othercellular components form a firm pellet at the bottom of a tube. RNA isprecipitated from the supernatant or filtrate with a water-solubleorganic solvent as previously described. The RNA precipitate is washedand may be dissolved in water, buffer, or formamide.

In one embodiment of the invention, a single reagent may be used ineither the phase separation method or the acidic phenol precipitationmethod. This dual use reagent comprises components of the reagent usedin the phase separation method and a 2× concentration of reagent used inthe acidic phenol precipitation method. As previously described, the pHfor the phase separation method may be between about pH 3.6 to below pH4.0, and the pH for the acidic phenol precipitation method may bebetween about pH 3.6 to about pH 5.5. In the dual use embodiment, a pHadjustment of the reagent is therefore necessary before switching fromone method to the other method. For example, the 2× reagent for theacidic phenol precipitation method at pH 4.2 must be further acidifiedto a pH below 4.0 before use in the phase separation method.

The inventive phase separation method may be used for specimenscontaining high amounts of fats, such as fat tissue and certain tumor orneoplastic tissues. Samples with a high level of contaminants, such asplants and fat-containing tissues, may be processed by the dual useprocedure, which combines the acidic phenol precipitation method and thephase separation method. In one embodiment, a biological sample ishomogenized in 2× acidic phenol precipitation reagent. The homogenate isthen diluted with water to approach the concentration range of theacidic phenol precipitation reagent (that is, about 3%^(w/w) phenol toless than 30%^(w/w) phenol). After dilution, precipitated DNA, proteinsand other cellular components are removed by sedimentation orfiltration. The resulting supernatant or filtrate is collected and mixedwith a phase separation solvent. In one embodiment, 0.05 volume to 0.01volume of a phase separation solvent is added per one volume of thesupernatant. The phase separation solvent or mix of solvents is at leastone hydrophobic solvent including, but not limited to, caprolactone,ethylene glycol diacetate, polyethylene glycol dibenzoate, as well assolvents used for the phase separation method. The mixture iscentrifuged to obtain a top aqueous phase, an interphase, and an organicphase. The aqueous phase containing RNA is collected and mixed with onevolume of a lower alcohol to precipitate RNA. The precipitated RNA iswashed and dissolved in water, buffer, or formamide.

The methods providing purified RNA based on the inventive acidic phenolsolutions are useful for gene expression profiling with RT-PCR basedmicroarrays used in biotechnology, molecular biology and clinicalapplications. This can be exemplified by the detection of specific geneexpression patterns in cancer cells and other type of pathologicalspecimens.

Selective RNA Precipitation Using Low Volume Organic Solvent

As previously described, RNA may be precipitated from the aqueous phasein the inventive phase separation method, and from the inventive acidicphenol composition. In each case, RNA is precipitated by adding aboutone volume of an organic solvent to approach a final organic solventconcentration of about 50%^(w/w). However, in some sample preparations,one volume of organic solvent co-precipitates polysaccharides andproteins, such as proteoglycans, together with RNA. Previously, to avoidco-precipitation of contaminants, one method modified the single-stepRNA purification method by employing 25%^(w/w) alcohol in the presenceof 0.9 M sodium ions to precipitate RNA (Chomczynski, 1995). Anothermethod treated a phenol-free chaotrope solution with 13%^(w/w) to23%^(w/w) of an organic solvent, with the pH of the solution remainingwithin the range of pH 6 to pH 7.5 to precipitate RNA.

In the present inventive method, substantially pure RNA is precipitatedfrom phenol-chaotrope solutions by adding an organic solvent or mix ofsolvents to achieve a final concentration of about 10%^(w/w) to about40%^(w/w) organic solvent. The organic solvent(s) may be acetone,tetramethylene sulfone, lower alcohols, glycols, polyalcohols, acetone,ethyleneglycol diacetate, and/or methyl sulfoxide. This method providessubstantially pure RNA when precipitating RNA from either the aqueousphase in the phase separation method, or in the acidic phenolprecipitation method from the DNA- and protein-free supernatant. Themethod does not require adding salts to a phenol-chaotrope solution.

It was unexpected that substantially pure RNA precipitated from aphenol-chaotrope solution at about 10%^(w/w) to about 40%^(w/w)concentration of an organic solvent without supplementing the solutionwith salt, as was earlier suggested (Chomczynski 1995). In fact, addingsalt along with alcohol decreased the purity of the isolated RNA. Thefinding that 10%^(w/w) to 40%^(w/w) alcohol alone precipitated RNA fromthe phenol-chaotrope solution was also contrary to a report where DNAprecipitated and RNA remained in a soluble form by adding 0.3 volume ofalcohol (final concentration 24%^(w/w)) to the phenol-chaotrope solution(Siebert, 1993).

In one embodiment, the final concentration of organic solvent(s) in thecomposition is from about 20%^(w/w) to about 25%^(w/w). In anotherembodiment, the final concentration of organic solvent(s) in thecomposition is from about 10% to about 40%. The pH of thephenol-chaotrope solution may range from about pH 2.0 to about pH 9.0.In one embodiment, the pH of the phenol-chaotrope solution ranges fromabout pH 3.5 to about pH 5.0.

Organic solvents at concentrations from about 10%^(w/w) to about40%^(w/w) precipitate RNA molecules greater than about 200 bases,considered as higher molecular weight RNA. RNA fragments less than about200 bases, along with polysaccharides and proteoglycans, remain insolution. Following precipitation of higher molecular weight RNA, thesmaller molecular weight RNA can be recovered from the solution byprecipitating with an additional amount of organic solvent to approach afinal concentration of about 50%^(w/w) or higher, for example, to about90%^(w/w).

The inventive method, whereby RNA is precipitated using about 10%^(w/w)to about 40%^(w/w) of an organic solvent, can also be used to decreasethe amount of contaminating DNA in RNA preparations. This selectiveprecipitation of RNA is effective only when a small amount ofcontaminating DNA is present in the solutions, for example, less than 10ng DNA per 1 μg RNA. Precipitating RNA using about 10%^(w/w) to about40%^(w/w) of an organic solvent also improves the quality of RNAisolated, such that a high yield of substantially pure and undegradedRNA is obtained, in accord with the method described in my previous '155and '994 patents.

Acidic RNA Precipitation from Salt-Containing Solutions

Substantially pure RNA, along with DNA, may be obtained from aqueoussolutions containing salts by pH-dependent precipitation of RNA at a pHbelow about 3.3. Precipitating RNA from salt solutions at an acidic pHis contrary to that reported in U.S. Pat. No. 5,973,137. The '137 patentdiscloses that, in solutions at pH below 6, non-chaotropic saltsprecipitate DNA, while RNA stays in a soluble form.

In the inventive method, a buffer acid is added to the RNA solution inan amount sufficient to result in a pH of 3.3 or lower. In oneembodiment, the resulting pH is in the range from about pH 3.0 to aboutpH 2.7. The acid may be an organic acid or an inorganic acid. The acidmay be hydrochloric acid, phosphoric acid, acetic acid, and/or lacticacid. In one embodiment, salts in the composition may be guanidinesalts.

This embodiment may be incorporated into either the inventive phaseseparation method and/or the inventive acidic phenol precipitationmethod. For use in the acidic phenol precipitation method, acids orbuffers can be dissolved either in water or in organic solvents. Theacid selectively precipitates RNA, leaving polysaccharides and proteinin a soluble form. The volume of acid used to precipitate RNA is small,permitting a low sample volume during RNA isolation. In one embodiment,the volume of acid ranges from about 0.1%^(w/w) to about 25%^(w/w) ofthe volume of RNA solution.

The precipitation of RNA with a low amount of an organic solvent andwith acidic pH, described in the present invention, can also be used toimprove the quality of RNA using methods disclosed in my previous '155and '994 patents.

Treating an RNA-containing sample obtained by RNA precipitation with alow volume of an organic solvent or acidic precipitation of RNA fromsalt-containing solutions precipitates higher molecular weight RNA.Higher molecular weight RNA includes ribosomal RNA (rRNA, for example18S and 28S RNA) and messenger RNA (mRNA). The remaining lower molecularweight RNA is less than about 200 nucleotides, and includes transfer RNA(tRNA), 5S RNA, and small interfering RNA (siRNA) that regulates geneexpression. Lower molecular weight RNA is recovered from theabove-described solution by treatment with an additional volume of anorganic solvent. In one embodiment, the sample is treated with onevolume of a lower alcohol, for example, methanol, ethanol, propanol,etc. to precipitate low molecular weight RNA.

Exemplary solutions and methods of the present invention are describedin the following working Examples.

EXAMPLE 1

Phase Separation Isolation of RNA from Rat Liver

In one embodiment, the following composition was used for phaseseparation of RNA: 4 M guanidine thiocyanate, 0.2 M ammoniumthiocyanate, 5%^(w/w) glycerol, 40%^(w/w) phenol, 0.1%^(w/w) sarcosine,10 mM sodium citrate, and 0.1 M sodium acetate buffer, pH 3.8.

Rat liver (38 mg) was homogenized in 1.5 ml of the above composition.Thereafter, 0.15 ml of bromochloropropane was added to the homogenate.The resulting mixture was shaken and sedimented for fifteen minutes at4° C. at 12,000×g. Following sedimentation, an aqueous phase, aninterphase, and a lower organic phase formed. RNA sequestered into theaqueous phase, while DNA and proteins sequestered into the interphaseand organic phase.

RNA was precipitated from the aqueous phase by adding 0.75 ml ofisopropanol. The RNA precipitate was centrifuged for five minutes at10,000×g. The resulting pellet was washed with 0.75 ml of 75%^(w/w)ethanol and centrifuged for five minutes at 10,000×g. The final RNApellet was dissolved in water and the RNA concentration was determinedspectrophotometrically at A_(260/280) by methods known to one skilled inthe art.

The yield of RNA was 0.22 mg. The A_(260/280) ratio was 1.76, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using primers for glyceraldehydes3-phosphate dehydrogenase (GAPDH), actin, and c-fos genes. Reversetranscription was performed using Superscript transcriptase fromInvitrogen (Carlsbad Calif.) and PCR was performed using Taq DNApolymerase from Sigma (St. Louis Mo.). RT-PCR products were analyzed ona 1% agarose-ethidium bromide gel. No DNA was detected in the isolatedRNA in the absence of reverse transcription. Northern blot analysis ofthe isolated RNA was performed using 1%-formaldehyde-agarose gels andtransferred to a nylon membrane. Ethidium bromide and methylene bluestaining showed undegraded ribosomal bands. Hybridization withbiotin-labeled probes showed undegraded bands of mRNA for GAPDH, actin,and c-fos.

EXAMPLE 2

Phase Separation Isolation of RNA from Human Blood

Human blood (0.5 ml) was mixed with 75 μl of glacial acetic acid and 5ml of the composition described in Example 1. Thereafter, 0.5 ml ofbromochloropropane was added to the mixture. The mixture was shaken andsedimented for fifteen minutes at 4° C. at 12,000×g. Followingsedimentation, the mixture formed an aqueous phase, an interphase, and alower organic phase. RNA sequestered into the aqueous phase, while DNAand proteins sequestered into the interphase and organic phase.

RNA was precipitated from the aqueous phase by adding 1.25 ml ofisopropanol. The RNA precipitate was centrifuged for five minutes at12,000×g. The resulting pellet was washed with five ml of 75% ethanoland centrifuged for five minutes at 10,000×g. The final RNA pellet wasdissolved in water and the RNA concentration was determinedspectrophotometrically at A_(260/280) by methods known to one skilled inthe art.

The yield of RNA was 18.9 μg. The A_(260/280) ratio was 1.70, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using GAPDH primers. No DNAcontamination was detected by PCR of the isolated RNA without reversetranscription. Northern blot analysis of the isolated RNA showedundegraded bands of ribosomal RNA and an undegraded band of GAPDH mRNA.

EXAMPLE 3

Isolation of RNA by Phase Separation and Acidified Bromochloropropane

Rat spleen (21mg) was homogenized in 1 ml of an aqueous solutioncontaining 3.5 M guanidine thiocyanate, 50 mM potassium acetate,43%^(w/w) phenol, 0.1% Triton X-100, pH 4.1. The homogenate wascentrifuged at 12,000×g for ten minutes to remove the bulk of DNA andparticulates. The clear homogenate was mixed with 0.1 ml ofbromochloropropane containing 14%^(w/w) acetic acid. The resulting pH ofthe mixture was pH 3.7. The mixture was shaken and sedimented for tenminutes at 4° C. at 12,000×g. Following sedimentation, the mixtureformed an aqueous phase, an interphase, and a lower organic phase. RNAsequestered into the aqueous phase, while DNA and proteins sequesteredinto the interphase and organic phase.

RNA was selectively precipitated from the aqueous phase by adding 0.5 mlethanol. The precipitated RNA was sedimented for five minutes at10,000×g, then washed with 75%^(w/w) ethanol and sedimented for fiveminutes at 10,000×g. The final RNA pellet was dissolved in water and theRNA concentration was determined spectrophotometrically at A_(260/280)by methods known to one skilled in the art.

The yield of RNA was 77 μl. The A_(260/280) ratio was 1.74, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using GAPDH primers and no DNAcontamination was detected. Northern blot analysis of the isolated RNAshowed undegraded bands of ribosomal RNA and an undegraded band of GAPDHmRNA.

EXAMPLE 4

Isolation of RNA by Phase Separation and Homogenization in a Phenol-FreeChaotrope Solution

Rat skeletal muscle (29 mg) was homogenized in 0.5 ml of an aqueoussolution of 3 M guanidine thiocyanate and 5 mM sodium acetate. Thehomogenate was mixed with 0.5 ml of phenol and 0.1 M sodium acetatebuffer, pH 3.7. The resulting mixture was shaken with 0.1 ml ofbromochloropropane and sedimented for fifteen minutes at 4° C. at12,000×g. Following sedimentation, the mixture formed an aqueous phase,an interphase, and a lower organic phase. RNA sequestered into theaqueous phase, while DNA and proteins sequestered into the interphaseand organic phase.

RNA was selectively precipitated from the aqueous phase by adding 0.5 mlof an aqueous solution containing 50%^(w/w) ethanol. The RNA precipitatewas washed, treated, and assayed as described in Example 1.

The yield of RNA was 16 μg. The A_(260/280) ratio was 1.70, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using GAPDH primers and no DNAcontamination was detected. Northern blot analysis of the isolated RNAshowed undegraded bands of ribosomal RNA and an undegraded band of GAPDHmRNA.

EXAMPLE 5

Isolation of RNA by Phase Separation with Homogenization in 1%^(w/w)Sodium Dodecyl Sulfate

A primary culture of human fibroblast cells (Clonetics, San DiegoCalif.) grown in a 25 cm² plastic bottle was overlaid with 1.5 ml of asolution containing 1%^(w/w) sodium dodecyl sulfate and 10 mM sodiumcitrate, pH 7.0, supplemented with 50 μg/ml proteinase K. The resultingcell solution was incubated for one hour at room temperature (about 20°C.), transferred to a centrifuge tube, and mixed with 1.5 ml of acidicphenol containing 12%^(w/w) water and 100 mM sodium acetate, pH 3.7.After centrifugation for fifteen minutes at 4° C., the mixture formed anaqueous phase, an interphase, and an organic phase. Following phaseseparation, RNA sequestered into the aqueous phase, while DNA andproteins sequestered into the interphase and organic phase,respectively.

RNA was precipitated from the aqueous phase by adding 0.75 ml of ethanoland sedimenting for five minutes at 10,000×g. The RNA pellet was washedwith 75% ethanol, centrifuged for five minutes at 10,000×g, anddissolved in water.

The yield of RNA was 18 μg. The A_(260/280) ratio was 1.71, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using GAPDH primers and no DNAcontamination was detected. Northern blot analysis of the isolated RNAshowed undegraded bands of ribosomal RNA and an undegraded band of GAPDHmRNA.

EXAMPLE 6

Isolation of RNA by Acidic Phenol Precipitation

In one embodiment, the following composition was used for acidic phenolprecipitation of RNA: 20%^(w/w) phenol, 2 M guanidine thiocyanate, 15 mMsodium citrate, 0.1 M lithium chloride, 0.05%^(w/w) sarcosine,1.5%^(w/w) glycerol, and sodium acetate buffer, pH 4.2. Rat liver (52mg) was homogenized in 1 ml of this composition. The homogenate wascentrifuged at 10,000×g for five minutes at room temperature (about 20°C.) to remove precipitated DNA, protein, and cellular components.

The resulting supernatant was transferred to a clean tube and was mixedwith 1 ml of ethanol to precipitate RNA. Precipitated RNA was sedimentedat 10,000×g for five minutes, washed with 75%^(w/w) ethanol, anddissolved in water.

The yield of RNA was 187 μg. The A_(260/280) ratio was 1.74, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using GAPDH primers and no DNAcontamination was detected. Northern blot analysis of the isolated RNAshowed undegraded bands of ribosomal RNA and an undegraded band of GAPDHmRNA.

EXAMPLE 7

Isolation of RNA by Acidic Phenol Precipitation using Two TimesConcentrated Reagent

Rat liver (47 mg) was homogenized in 1 ml of the reagent described inExample 6 at two times the concentration indicated in Example 6. Theconcentrated reagent contained additionally 1%^(w/w) phenylethanol. Thehomogenate was mixed with 1 ml water to form the precipitating reagent.

The precipitated DNA, proteins and other cellular components weresedimented by centrifugation at 10,000×g for five minutes at roomtemperature (about 20° C.). The resulting supernatant was transferred toa clean tube and mixed with 1 ml ethanol to precipitate RNA.Precipitated RNA was sedimented at 10,000×g for 5 minutes, washed with75%^(w/w) ethanol, and dissolved in water.

The yield of RNA was 178 μg. The A_(260/280) ratio was 1.77, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using GAPDH primers and no DNAcontamination was detected. Northern blot analysis of the isolated RNAshowed undegraded bands of ribosomal RNA and an undegraded band of GAPDHmRNA.

EXAMPLE 8

Isolation of RNA by the Inventive Two-Step Procedure

Rat brain (61 mg) was homogenized in 1 ml of the two times concentratedreagent described in Example 7. The homogenate was mixed with 1 ml waterand the resulting mixture was centrifuged at 10,000×g for five minutesat room temperature (about 20° C.) to remove precipitated DNA, protein,and cellular components. The supernatant was transferred to a clean tubeand mixed with 0.05 ml of bromochloropropane. The mixture wascentrifuged and separated into a top aqueous phase, an interphase, andan organic phase.

The aqueous phase containing RNA was transferred to a fresh tube and wasacidified to pH 2.9 with 5 M lactic acid in isopropanol. The RNAprecipitate was sedimented at 10,000×g for five minutes, washed with75%^(w/w) ethanol, and dissolved in water.

The yield of RNA was 33 μg. The A_(260/280) ratio was 1.77, whichindicated the lack of protein contamination. The isolated RNA wassuccessfully utilized for RT-PCR using GAPDH primers and no DNAcontamination was detected. Northern blot analysis of the isolated RNAshowed undegraded bands of ribosomal RNA and an undegraded band of GAPDHmRNA.

Other variations or embodiments of the invention will also be apparentto one of ordinary skill in the art from the above description andexamples. Thus, the forgoing embodiments are not to be construed aslimiting the scope of this invention.

1. A set of reagents for isolating purified RNA from a biological samplecomprising a biological sample and a RNA isolating reagent comprisingphenol that when added to the biological sample is at a finalconcentration ranging from about 10%^(w/w) to about 60%^(w/w) and abuffer sufficient to acidify and maintain a pH of the biological samplein the range from about pH 3.6 to below pH 4.0.
 2. A set of reagents forisolating purified RNA from a biological sample comprising a biologicalsample and a RNA isolating reagent comprising phenol that when added tothe biological sample is at a final concentration ranging from about3%^(w/w) to less than 30%^(w/w), and a buffer sufficient to acidify andmaintain a pH of the biological sample in the range from about pH 3.6 toabout pH 5.5.
 3. The composition according to claim 1 where the bufferis selected from at least one of acetate, citrate, phosphate, phthalate,tartrate, lactate, or mixtures thereof.
 4. The composition according toclaim 1 further comprising at least one ribonuclease inhibitor.
 5. Thecomposition of claim 4 wherein the ribonuclease inhibitor is selectedfrom at least one of proteinase K, ribonuclease inhibitor from humanplacenta, vanadyl ribonucleoside complex, chaotropic salts, or mixturesthereof.
 6. The composition of claim 5 wherein the chaotropic salts areselected from at least one of urea salts, guanidine salts, or mixturesthereof.
 7. The composition of claim 6 wherein the guanidine salts areselected from at least one of guanidine thiocyanate or guanidinehydrochloride at a final concentration in the range of about 0.5 M toabout 6 M.
 8. The composition according to claim 1 further comprising adetergent.
 9. The composition of claim 8 wherein the detergent isselected from at least one of sarcosine, polyoxyethylenesorbitan, adodecylsulfate salt, or mixtures thereof.
 10. The composition accordingto claim 1 further comprising an inorganic or organic salt and achelating agent.
 11. The composition of claim 10 wherein the inorganicor organic salt is selected from at least one of chlorides, phosphates,bromates, acetates, citrates, phthalates, tartrates, lactates, orthiocyanates of sodium, potassium, lithium or ammonium.
 12. Thecomposition of claim 10 wherein the chelating agent is selected from atleast one of citrates, ethylenediamine tetraacetic salts, or mixturesthereof.
 13. The composition according to claim 1 further comprisingphenol derivatives selected from at least one of phenylethanol,propylene phenoxytol, thymol, butylphenol, or mixtures thereof at afinal concentration up to about 5%^(w/w).
 14. The composition accordingto claim 1 further comprising phenol solubilizers selected from at leastone of polyalcohols, monoalcohols, and guanidine salts.
 15. Thecomposition of claim 1 further comprising at least one organic compoundin a concentration ranging from about 1%^(w/w) to about 5%^(w/w)sufficient to increase the density of the composition.
 16. Thecomposition of claim 15 wherein the organic compound is selected from atleast one of cyclohexyl bromide, dibromopropane, dichlorobenzoic acid,and mixtures thereof.
 17. The composition according to claim 2 where thebuffer is selected from at least one of acetate, citrate, phosphate,phthalate, tartrate, lactate, or mixtures thereof.
 18. The compositionaccording to claim 2 further comprising at least one ribonucleaseinhibitor.
 19. The composition of claim 18 wherein the ribonucleaseinhibitor is selected from at least one of proteinase K, ribonucleaseinhibitor from human placenta, vanadyl ribonucleoside complex,chaotropic salts, or mixtures thereof.
 20. The composition of claim 19wherein the chaotropic salts are selected from at least one of ureasalts, guanidine salts, or mixtures thereof.
 21. The composition ofclaim 20 wherein the guanidine salts are selected from at least one ofguanidine thiocyanate or guanidine hydrochloride at a finalconcentration in the range of about 0.5 M to about 6 M.
 22. Thecomposition according to claim 2 further comprising a detergent.
 23. Thecomposition of claim 22 wherein the detergent is selected from at leastone of sarcosine, polyoxyethylenesorbitan, a dodecylsulfate salt, ormixtures thereof.
 24. The composition according to claim 2 furthercomprising an inorganic or organic salt and a chelating agent.
 25. Thecomposition of claim 24 wherein the inorganic or organic salt isselected from at least one of chlorides, phosphates, bromates, acetates,citrates, phthalates, tartrates, lactates, or thiocyanates of sodium,potassium, lithium or ammonium.
 26. The composition of claim 24 whereinthe chelating agent is selected from at least one of citrates,ethylenediamine tetraacetic salts, or mixtures thereof.
 27. Thecomposition according to claim 2 further comprising phenol derivativesselected from at least one of phenylethanol, propylene phenoxytol,thymol, butylphenol, or mixtures thereof at a final concentration up toabout 5%^(w/w).
 28. The composition according to claim 2 furthercomprising phenol solubilizers selected from at least one ofpolyalcohols, monoalcohols, and guanidine salts.
 29. The composition ofclaim 2 further comprising at least one organic compound in aconcentration ranging from about 1%^(w/w) to about 5%^(w/w) sufficientto increase the density of the composition.
 30. The composition of claim29 wherein the organic compound is selected from at least one ofcyclohexyl bromide, dibromopropane, dichlorobenzoic acid, and mixturesthereof.
 31. The composition of claim 8 wherein the detergentconcentration is about 0.1%^(w/w) or less.